Cannabidiol Protects against the Reinstatement of Oxycodone-Induced Conditioned Place Preference in Adolescent Male but Not Female Rats: The Role of MOR and CB1R.

Cannabidiol (CBD), a phytocannabinoid, appeared to satisfy several criteria for a safe approach to preventing drug-taking behavior, including opioids. However, most successful preclinical and clinical results come from studies in adult males. We examined whether systemic injections of CBD (10 mg/kg, i.p.) during extinction of oxycodone (OXY, 3 mg/kg, i.p.) induced conditioned place preference (CPP) could attenuate the reinstatement of CPP brought about by OXY (1.5 mg/kg, i.p.) priming in adolescent rats of both sexes, and whether this effect is sex dependent. Accordingly, a priming dose of OXY produced reinstatement of the previously extinguished CPP in males and females. In both sexes, this effect was linked to locomotor sensitization that was blunted by CBD pretreatments. However, CBD was able to prevent the reinstatement of OXY-induced CPP only in adolescent males and this outcome was associated with an increased cannabinoid 1 receptor (CB1R) and a decreased mu opioid receptor (MOR) expression in the prefrontal cortex (PFC). The reinstatement of CCP in females was associated with a decreased MOR expression, but no changes were detected in CB1R in the hippocampus (HIP). Moreover, CBD administration during extinction significantly potentialized the reduced MOR expression in the PFC of males and showed a tendency to potentiate the reduced MOR in the HIP of females. Additionally, CBD reversed OXY-induced deficits of recognition memory only in males. These results suggest that CBD could reduce reinstatement to OXY seeking after a period of abstinence in adolescent male but not female rats. However, more investigation is required.

Social Interaction in Adolescent Rats with Neonatal Ethanol Exposure: Impact of Sex and CE-123, a Selective Dopamine Reuptake Inhibitor.

Children with fetal alcohol spectrum disorders (FASDs) demonstrate deficits in social functioning that contribute to early withdrawal from school and delinquency, as well as the development of anxiety and depression. Dopamine is involved in reward, motivation, and social behavior. Thus, we evaluated whether neonatal ethanol exposure (in an animal model of FASDs) has an impact on social recognition memory using the three-chamber social novelty discrimination test during early and middle adolescence in male and female rats, and whether the modafinil analog, the novel atypical dopamine reuptake inhibitor CE-123, can modify this effect. Our study shows that male and female rats neonatally exposed to ethanol exhibited sex- and age-dependent deficits in social novelty discrimination in early (male) and middle (female) adolescence. These deficits were specific to the social domain and not simply due to more general deficits in learning and memory because these animals did not exhibit changes in short-term recognition memory in the novel object recognition task. Furthermore, early-adolescent male rats that were neonatally exposed to ethanol did not show changes in the anxiety index but demonstrated an increase in locomotor activity. Chronic treatment with CE-123, however, prevented the appearance of these social deficits. In the hippocampus of adolescent rats, CE-123 increased BDNF and decreased its signal transduction TrkB receptor expression level in ethanol-exposed animals during development, suggesting an increase in neuroplasticity. Thus, selective dopamine reuptake inhibitors, such as CE-123, represent interesting drug candidates for the treatment of deficits in social behavior in adolescent individuals with FASDs.

Neonatal Maternal Separation Induces Sexual Dimorphism in Brain Development: The Influence on Amino Acid Levels and Cognitive Disorders.

Repeated maternal separation (MS) is a useful experimental model in rodents for studying the long-term influence of early-life stress on brain neurophysiology. In our work, we assessed the effect of repeated MS (postnatal day (PND)1-21, 180 min/day) on the postnatal development of rat brain regions involved in memory using proton magnetic resonance spectroscopy (1HMRS) for tissue volume and the level of amino acids such as glutamate, aspartate, glutamine, glycine and gamma-aminobutyric acid (GABA) in the hippocampus. We assessed whether these effects are sex dependent. We also use novel object recognition (NOR) task to examine the effect of MS on memory and the effect of ethanol on it. Finally, we attempted to ameliorate postnatal stress-induced memory deficits by using VU-29, a positive allosteric modulator (PAM) of the metabotropic glutamate type 5 (mGlu5) receptor. In males, we noted deficits in the levels of glutamate, glycine and glutamine and increases in GABA in the hippocampus. In addition, the values of perirhinal cortex, prefrontal cortex and insular cortex and CA3 were decreased in these animals. MS females, in contrast, demonstrated significant increase in glutamate levels and decrease in GABA levels in the hippocampus. Here, the CA1 values alone were increased. VU-29 administration ameliorated these cognitive deficits. Thus, MS stress disturbs amino acids levels mainly in the hippocampus of adult male rats, and enhancement of glutamate neurotransmission reversed recognition memory deficits in these animals.

Impact of Mephedrone on Fear Memory in Adolescent Rats: Involvement of Matrix Metalloproteinase-9 (MMP-9) and N-Methyl-D-aspartate (NMDA) Receptor.

Treatment of Post-Traumatic Stress Disorder (PTSD) is complicated by the presence of drug use disorder comorbidity. Here, we examine whether conditioned fear (PTSD model) modifies the rewarding effect of mephedrone and if repeated mephedrone injections have impact on trauma-related behaviors (fear sensitization, extinction, and recall of the fear reaction). We also analyzed whether these trauma-induced changes were associated with exacerbation in metalloproteinase-9 (MMP-9) and the GluN2A and GluN2B subunits of N-methyl-D-aspartate (NMDA) glutamate receptor expression in such brain structures as the hippocampus and basolateral amygdala. Male adolescent rats underwent trauma exposure (1.5 mA footshock), followed 7 days later by a conditioned place preference training with mephedrone. Next, the post-conditioning test was performed. Fear sensitization, conditioned fear, anxiety-like behavior, extinction acquisition and relapse were then assessed to evaluate behavioral changes. MMP-9, GluN2A and GluN2B were subsequently measured. Trauma-exposed rats subjected to mephedrone treatment acquired a strong place preference and exhibited impairment in fear extinction and reinstatement. Mephedrone had no effect on trauma-induced MMP-9 level in the basolateral amygdala, but decreased it in the hippocampus. GluN2B expression was decreased in the hippocampus, but increased in the basolateral amygdala of mephedrone-treated stressed rats. These data suggest that the modification of the hippocampus and basolateral amygdala due to mephedrone use can induce fear memory impairment and drug seeking behavior in adolescent male rats.

Novel Dopamine Transporter Inhibitor, CE-123, Ameliorates Spatial Memory Deficits Induced by Maternal Separation in Adolescent Rats: Impact of Sex.

Maternal separation (MS) is a key contributor to neurodevelopmental disorders, including learning disabilities. To test the hypothesis that dopamine signaling is a major factor in this, an atypical new dopamine transporter (DAT) inhibitor, CE-123, was assessed for its potential to counteract the MS-induced spatial learning and memory deficit in male and female rats. Hence, neonatal rats (postnatal day (PND)1 to 21) were exposed to MS (180 min/day). Next, the acquisition of spatial learning and memory (Barnes maze task) and the expression of dopamine D1 receptor, dopamine transporter (DAT), and the neuronal GTPase, RIT2, which binds DAT in the vehicle-treated rats were evaluated in the prefrontal cortex and hippocampus in the adolescent animals. The results show that MS impairs the acquisition of spatial learning and memory in rats, with a more severe effect in females. Moreover, the MS induced upregulation of DAT and dopamine D1 receptors expression in the prefrontal cortex and hippocampus in adolescent rats. Regarding RIT2, the expression was decreased in the hippocampus for both the males and females, however, in the prefrontal cortex, reduction was found only in the females, suggesting that there are region-specific differences in DAT endocytic trafficking. CE-123 ameliorated the behavioral deficits associated with MS. Furthermore, it decreased the MS-induced upregulation of D1 receptor expression level in the hippocampus. These effects were more noted in females. Overall, CE-123, an atypical DAT inhibitor, is able to restore cognitive impairment and dopamine signaling in adolescent rats exposed to MS-with more evident effect in females than males.

Maternal Separation Alters Ethanol Drinking and Reversal Learning Processes in Adolescent Rats: The Impact of Sex and Glycine Transporter Type 1 (GlyT1) Inhibitor.

Adverse early life experiences are associated with an enhanced risk for mental and physical health problems, including substance abuse. Despite clinical evidence, the mechanisms underlying these relationships are not fully understood. Maternal separation (MS) is a commonly used animal model of early neglect. The aim of the current study is to determine whether the N-methyl-D-aspartate receptor (NMDAR)/glycine sites are involved in vulnerability to alcohol consumption (two-bottle choice paradigm) and reversal learning deficits (Barnes maze task) in adolescent rats subjected to the MS procedure and whether these effects are sex dependent. By using ELISA, we evaluated MS-induced changes in the NMDAR subunits (GluN1, GluN2A, GluN2B) expression, especially in the glycine-binding subunit, GluN1, in the prefrontal cortex (PFC) and ventral striatum (vSTR) of male/female rats. Next, we investigated whether Org 24598, a glycine transporter 1 (GlyT1) inhibitor, was able to modify ethanol drinking in adolescent and adult male/female rats with prior MS experience and reversal learning in the Barnes maze task. Our findings revealed that adolescent MS female rats consumed more alcohol which may be associated with a substantial increase in GluN1 subunit of NMDAR in the PFC and vSTR. Org 24598 decreased ethanol intake in both sexes with a more pronounced decrease in ethanol consumption in adolescent female rats. Furthermore, MS showed deficits in reversal learning in both sexes. Org 24598 ameliorated reversal learning deficits, and this effect was reversed by the NMDAR/glycine site inhibitor, L-701,324. Collectively, our results suggest that NMDAR/glycine sites might be targeted in the treatment of alcohol abuse in adolescents with early MS, especially females.

Alteration of Ethanol Reward by Prior Mephedrone Exposure: The Role of Age and Matrix Metalloproteinase-9 (MMP-9).

Mephedrone, a synthetic cathinone, is widely abused by adolescents and young adults. The aim of this study was to determine: (i) whether prior mephedrone exposure would alter ethanol reward and (ii) whether age and matrix metalloproteinase-9 (MMP-9) are important in this regard. In our research, male Wistar rats at postnatal day 30 (PND30) received mephedrone at the dose of 10 mg/kg, i.p., 3 times a day for 7 days. To clarify the role of MMP-9 in the mephedrone effects, one mephedrone-treated group received minocycline, as an MMP-9 antagonist. Animals were then assigned to conditioned place preference (CPP) procedure at PND38 (adolescent) or at PND69 (adult). After the CPP test (PND48/79), expression of dopamine D1 receptors (D1R), Cav1.2 (a subtype of L-type calcium channels), and MMP-9 was quantified in the rat ventral striatum (vSTR). The influence of mephedrone administration on the N-methyl-D-aspartate glutamate receptors (NMDAR) subunits (GluN1, GluN2A, and GluN2B) was then assessed in the vSTR of adult rats (only). These results indicate that, in contrast with adolescent rats, adult rats with prior mephedrone administration appear to be more sensitive to the ethanol effect in the CPP test under the drug-free state. The mephedrone effect in adult rats was associated with upregulation of D1R, NMDAR/GluN2B, MMP-9, and Cav1.2 signaling. MMP-9 appears to contribute to these changes in proteins expression because minocycline pretreatment blocked mephedrone-evoked sensitivity to ethanol reward. Thus, our results suggest that prior mephedrone exposure differentially alters ethanol reward in adolescent and adult rats.

Memantine Prevents the WIN 55,212-2 Evoked Cross-Priming of Ethanol-Induced Conditioned Place Preference (CPP).

The activation of the endocannabinoid system controls the release of many neurotransmitters involved in the brain reward pathways, including glutamate. Both endocannabinoid and glutamate systems are crucial for alcohol relapse. In the present study, we hypothesize that N-methyl-D-aspartate (NMDA) glutamate receptors regulate the ability of a priming dose of WIN 55,212-2 to cross-reinstate ethanol-induced conditioned place preference (CPP). To test this hypothesis, ethanol-induced (1.0 g/kg, 10% w/v, i.p.) CPP (unbiased method) was established using male adult Wistar rats. After CPP extinction, one group of animals received WIN 55,212-2 (1.0 and 2.0 mg/kg, i.p.), the cannabinoid receptor 1 (CB1) agonist, or ethanol, and the other group received memantine (3.0 or 10 mg/kg, i.p.), the NMDA antagonist and WIN 55,212-2 on the reinstatement day. Our results showed that a priming injection of WIN 55,212-2 (2.0 mg/kg, i.p.) reinstated (cross-reinstated) ethanol-induced CPP with similar efficacy to ethanol. Memantine (3.0 or 10 mg/kg, i.p.) pretreatment blocked this WIN 55,212-2 effect. Furthermore, our experiments indicated that ethanol withdrawal (7 days withdrawal after 10 days ethanol administration) down-regulated the CNR1 (encoding CB1), GRIN1/2A (encoding GluN1 and GluN2A subunit of the NMDA receptor) genes expression in the prefrontal cortex and dorsal striatum, but up-regulated these in the hippocampus, confirming the involvement of these receptors in ethanol rewarding effects. Thus, our results show that the endocannabinoid system is involved in the motivational properties of ethanol, and glutamate may control cannabinoid induced relapse into ethanol seeking behavior.

CE-123, a novel dopamine transporter inhibitor, attenuates locomotor hyperactivity and improves cognitive functions in rat model of fetal alcohol spectrum disorders.

Perinatal alcohol exposure can lead to fetal alcohol spectrum disorders (FASD), usually first diagnosed in childhood, that are characterized by hyperactivity, impulsivity and learning and memory disability, among others. To test the hypothesis that dopamine signaling is one of the main factors underlying these impairments, a new atypical dopamine transporter (DAT) inhibitor, CE-123 (1, 3 or 10 mg/kg) was assessed for its potential to overcome the ethanol-induced behavioral effects in a rat model of FASD. In the present study, neonatal rats were exposed to alcohol intubations across the neonatal period (postnatal day (PND)4-9, the third trimester equivalent of human gestation) and, after weaning, the animals (male rats) were assigned randomly to three groups. The first group was tested at PND21 (hyperactivity test). A second group was tested at PND45 (anxiety test), at PND47 (locomotor activity test), at PND49 (spatial cognitive test in the Barnes maze) and PND50 (reversal learning in the Barnes maze). The third group was tested at PND50 (dopamine receptor mRNA expression). Our results support the hypothesis that dopamine signaling is associated with FASD because the dopamine (D1, D2 and D5) receptor mRNA expression was altered in the striatum, hippocampus and prefrontal cortex in adult rats exposed to ethanol during neonatal period. CE-123 (3 and 10 mg/kg) inhibited the hyperactivity and ameliorated (10 mg/kg) the impairment of reversal learning in alcohol-exposed rats. Thus, these findings provide support that CE-123 may be a useful intervention for same of the deficits associated with neonatal ethanol exposure.

Rapamycin Improves Spatial Learning Deficits, Vulnerability to Alcohol Addiction and Altered Expression of the GluN2B Subunit of the NMDA Receptor in Adult Rats Exposed to Ethanol during the Neonatal Period.

Ethanol exposure during pregnancy alters the mammalian target of rapamycin (mTOR) signaling pathway in the fetal brain. Hence, in adult rats exposed to ethanol during the neonatal period, we investigated the influence of rapamycin, an mTOR Complex 1 (mTORC1) inhibitor, on deficits in spatial memory and reversal learning in the Barnes maze task, as well as the ethanol-induced rewarding effects (1.0 or 1.5 g/kg) using the conditioning place preference (CPP) paradigm. Rapamycin (3 and 10 mg/kg) was given before intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4-9 (an equivalent to the third trimester of human pregnancy). Spatial memory/reversal learning and rewarding ethanol effect were evaluated in adult (PND60-70) rats. Additionally, the impact of rapamycin pre-treatment on the expression of the GluN2B subunit of NMDA receptor in the brain was assessed in adult rats. Our results show that neonatal ethanol exposure induced deficits in spatial memory and reversal learning in adulthood, but the reversal learning outcome may have been due to spatial learning impairments rather than cognitive flexibility impairments. Furthermore, in adulthood the ethanol treated rats were also more sensitive to the rewarding effect of ethanol than the control group. Rapamycin prevented the neonatal effect of ethanol and normalized the GluN2B down-regulation in the hippocampus and the prefrontal cortex, as well as normalized this subunit's up-regulation in the striatum of adult rats. Our results suggest that rapamycin and related drugs may hold promise as a preventive therapy for fetal alcohol spectrum disorders.

Effects of Mephedrone and Amphetamine Exposure during Adolescence on Spatial Memory in Adulthood: Behavioral and Neurochemical Analysis.

A synthetic cathinone, mephedrone is widely abused by adolescents and young adults. Despite its widespread use, little is known regarding its long-term effects on cognitive function. Therefore, we assessed, for the first time, whether (A) repeated mephedrone (30 mg/kg, i.p., 10 days, once a day) exposure during adolescence (PND 40) induces deleterious effects on spatial memory and reversal learning (Barnes maze task) in adult (PND 71-84) rats and whether (B) these effects were comparable to amphetamine (2.5 mg/kg, i.p.). Furthermore, the influence of these drugs on MMP-9, NMDA receptor subunits (GluN1, GluN2A/2B) and PSD-95 protein expression were assessed in adult rats. The drug effects were evaluated at doses that per se induce rewarding/reinforcing effects in rats. Our results showed deficits in spatial memory (delayed effect of amphetamine) and reversal learning in adult rats that received mephedrone/amphetamine in adolescence. However, the reversal learning impairment may actually have been due to spatial learning rather than cognitive flexibility impairments. Furthermore, mephedrone, but not amphetamine, enhanced with delayed onset, MMP-9 levels in the prefrontal cortex and the hippocampus. Mephedrone given during adolescence induced changes in MMP-9 level and up-regulation of the GluN2B-containing NMDA receptor (prefrontal cortex and hippocampus) in young adult (PND 63) and adult (PND 87) rats. Finally, in adult rats, PSD-95 expression was increased in the prefrontal cortex and decreased in the hippocampus. In contrast, in adult rats exposed to amphetamine in adolescence, GluN2A subunit and PSD-95 expression were decreased (down-regulated) in the hippocampus. Thus, in mephedrone-but not amphetamine-treated rats, the deleterious effects on spatial memory were associated with changes in MMP-9 level. Because the GluN2B-containing NMDA receptor dominates in adolescence, mephedrone seems to induce more harmful effects on cognition than amphetamine does during this period of life.

The kisspeptin derivative kissorphin reduces the acquisition, expression, and reinstatement of ethanol-induced conditioned place preference in rats.

Research has shown that opioids are involved in the rewarding effects of ethanol. Neuropeptide FF (NPFF) has been described as an anti-opioid peptide because, in many cases, it inhibits opioid and ethanol effects in rodents. Kissorphin (KSO) is a new peptide derived from kisspeptin-10 with structural similarities to NPFF. This peptide possesses NPFF-like biological activity in vitro. The aim of the current study was to investigate whether KSO (Tyr-Asn-Trp-Asn-Ser-Phe-NH2) influences the acquisition, expression, and reinstatement of ethanol-induced conditioned place preference (ethanol-CPP) in rats. The ethanol-CPP was established (conditioning for 5 days) by intraperitoneal (i.p.) administration of ethanol (1 g/kg, 20%, w/v) using an unbiased procedure. After that, one group of rats was used in final post-conditioning testing (expression of CPP) and the other group received a priming injection of ethanol after 10 days of extinction (reinstatement of CPP). Our experiments showed that KSO, given intravenously (i.v.) at the doses of 1, 3, and 10 nmol before every ethanol administration, inhibited the acquisition and, given acutely before the post-conditioning test or before the priming dose of ethanol, inhibited the expression and reinstatement of ethanol-CPP, respectively, in a dose-dependent manner. KSO given by itself neither induced place preference nor aversion and did not alter locomotor activity and coordination of rats. These results suggest that KSO can alter rewarding/motivational effects of ethanol. These data suggest this peptide possesses an anti-opioid character.